Intrahepatic bile acid retention in BALB/c-Abcb4-/--mice activates FXR-dependent rescue transporters at the basolateral and canalicular membrane
Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive obliteration of bile ducts, accumulation of bile acids and secondary biliary fibrosis. Abcb4 involves the transport of phospholipids from hepatocytes into bile across the canalicular membrane. Abcb4 knockout disturbes the excretion of phospholipids into bile leading to a more toxic bile composition and bile duct alterations resemble sclerosing cholangitis. Hence we analyzed hepatic bile acid transport in Abcb4–/– mice during the course of the disease.
Methods: Total serum bile acids were measured (TBA) by diazyme kit. Expression of bile acid transporters (Ntcp, Oatp, Bsep, Mrp3, Mrp4, Ostα), nuclear receptors (Fxr), hepatic nuclear factors (HNF4a, HNF1a) and bile acid rate limiting enzyme Cyp7a1 were analyzed by qRT-PCR in liver tissue of BALB/c-Abcb4 –/– mice. Quantification of Ntcp and Oatp protein was performed by Western blot and immunostaining.
Results: Enhanced serum bile acids were observed in Abcb4–/– compared to wild type mice (week (w) 8 vs. w 26). Ntcp and Oatp mRNA expression was significantly downregulated in liver of Abcb4 –/– up to 26w. Bsep and Ostα transcript levels were downregulated at 8w but upregulated after 26w indicating that Ostα plays a prominent role in the retrograde bile acid transport with increasing severity of cholestasis. Downregulation of FXR in 8w mice correlates with decreased expression of its target genes Bsep and Ostα and inversely with an increased negative target Cyp7a1, clearly demonstrating that FXR-dependent feedback regulation was gradually activated over time. In parallel to Ostα increased expression of the basolateral exporters Mrp3 and -4 could be observed. Immunohistology revealed unequal distribution of Ntcp and Oatp in 8w and 26w Abcb4 –/– mice. Oatp protein expression was reduced at 8w while Ntcp was downregulated not before 26w, demonstrating that bile acid uptake was gradually abrogated in parallel to the activation of escape transporters.
Conclusion: With increasing retention of intrahepatic bile acids in the chronically injured liver of BALB/c-Abcb4 –/–-mice FXR-dependent escape mechanisms are activated at both the basolateral and the canalicular membrane compartment to protect from hepatocellular injury.